vermicomposting of coal fly ash using epigeic and epi

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Vermicompostin

Laboratory of Applied Microbiology, Dep

Engineering, Center of Mining Environmen

School of Mines), Dhanbad, Jharkhand, Ind

+91 9471191352

Cite this: RSC Adv., 2017, 7, 4876

Received 25th November 2016Accepted 5th January 2017

DOI: 10.1039/c6ra27329g

www.rsc.org/advances

4876 | RSC Adv., 2017, 7, 4876–4890

g of coal fly ash using epigeic andepi-endogeic earthworm species: nutrientdynamics and metal remediation

Zeba Usmani, Vipin Kumar* and Sujeet Kumar Mritunjay

Huge amounts of coal fly ash (FA), produced during power generation, has led to several environmental

problems associated with metal pollution and the burden of its disposal. Vermicomposting has emerged

as a cost-effective technique in suitable management of FA. Two epigeic earthworm species: Eisenia

fetida, and Eudrilus eugeniae and one epi-endogeic species: Lumbricus rubellus were selected for

vermiremediation of coal FA. The investigation addresses the changes in the earthworm biomass,

number, nutrient content and metal content in the treatments of FA amended with cow-dung (CD) at

periodical intervals of vermicomposting. Metal (Cr, Cu, Cd, Zn, Ni and Pb) removal efficiency of the three

earthworm species has been observed. Bioaccumulation factor (BAF) and changes in the microbiological

fauna of the different mixtures were also witnessed. Earthworm and cocoon count showed an increase

in trend with duration of vermicomposting. The concentration of total nitrogen, phosphorous and

potassium increased in all the treatments. The maximum bacterial count (8.0 � 105 CFU g�1) and fungal

count (3.8 � 103 CFU g�1) were observed in FA + CD (1 : 3) mixture, comprising E. eugeniae. Significant

reduction (p < 0.05) in the metal concentration of the treatments along with subsequent increase in

metal content of earthworm tissues using Flame Atomic Absorption Spectrophotometer was observed.

The treatment FA + CD (1 : 3) gave the best results in terms of nutrient enhancement and metal removal.

Maximum metal reduction in the treatment, FA + CD (1 : 3) was 58.82% for Cr by E. fetida, 71.94% for Ni

by E. eugeniae, and 51.67% for Cu by L. rubellus. Highest BAF value was obtained for Ni (3.31) in E.

eugeniae driven treatment FA + CD (1 : 3). The maximum metallothionein production was observed in E.

eugeniae followed by E. fetida and L. rubellus.

1. Introduction

Rapid urbanization and industrialization have intensied thedemand of electricity all over the world. Power has been regar-ded as an engine of growth in most of the developing countries.Major source of electrical energy is coal based thermal powerplants, leading to hyperbolic use of coal, as the prime energyresource. Fly ash (FA) is a resultant of coal and lignitecombustion1 that enters the ue gas stream2 during electricitygeneration. FA is one of the most complex and anthropogenicmaterial, leading to major environmental problems like soil,water, air pollution and disruption of ecological cycles. Thegrowing energy demand would lead to various social, economicand environmental problems related to FA disposal. Thus, thereis an urgent and ongoing need to develop novel methods forproper utilization andmanagement of coal FA. Recycling of coal

artment of Environmental Science and

t, Indian Institute of Technology (Indian

ia. E-mail: [email protected]; Tel:

FA can be a good alternative than its disposal leading toeconomic and environmental benets.

The physico-chemical and elemental properties of coal FAdetermines its utilization in different sectors of the economy.FA has great potentiality in agriculture and is quite economicalto use as a soil amendment.3 FA is a good source of essentialplant nutrients like Ca, Mg, S, Si, Al, Fe, and Na that are bene-cial for plant growth4 but it consists of certain heavy elementslike Fe, Mn, As, Cr, Zn, Pb, Ni, Ba, Sr and V, some of which are ofenvironmental concern even at low concentrations.5 Thus, themajor constraints associated with the use of FA in agriculturalecosystem is the low availability of the nutrient elements basi-cally N, P, K and lower rate of degradation of FA aer applica-tion in soils.6 The other serious problem of utilizing FA foragricultural use is the predominance of heavy metals in thematerial and in soluble forms.7,8

Vermicomposting is an effective technique for mitigation ofmetals from FA. It is an environment-friendly and faster methodof producing organic rich fertilizer from waste materials.9

Earthworms are potent for rapid and efficient decomposition ofvarious industrial and organic wastes.10 They have the abilityto convert waste materials into mineralized forms as

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Table 1 Treatments and codes of different combinations of fly ashand cow-dung along with the species of earthworms (Eisenia fetida,Eudrilus eugeniae and Lumbricus rubellus)a

Code Treatments

Ef1 FA alone + Eisenia fetidaEf2 FA + CD (1 : 1) + E. fetidaEf3 FA + CD (1 : 3) + E. fetidaEf4 FA + CD (3 : 1) + E. fetidaEf5 CD alone + E. fetidaEe1 FA alone + Eudrilus eugeniaeEe2 FA + CD (1 : 1) + E. eugeniaeEe3 FA + CD (1 : 3) + E. eugeniaeEe4 FA + CD (3 : 1) + E. eugeniaeEe5 CD alone + E. eugeniaeLr1 FA alone + Lumbricus rubellusLr2 FA + CD (1 : 1) + L. rubellusLr3 FA + CD (1 : 3) + L. rubellusLr4 FA + CD (3 : 1) + L. rubellusLr5 CD alone + L. rubellus

a FA: y ash; CD: cow-dung; Ef: Eisenia fetida; Ee: Eudrilus eugeniae; Lr:Lumbricus rubellus.

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vermicompost. Earthworm species such as Eisenia fetida, Lum-bricus rubellus, and Eudrilus eugeniae have the potency tomechanically fragment waste material through their gizzard byincreasing the surface area and positively modifying the bio-logical activity during composting. E. fetida has shown its abilityin increasing bioavailability of major nutrient elements such asP and N in FA11,12 on one hand and minimizing the solubility ofheavy metals on the other.13 The efficiency of E. eugeniae and L.rubellus in rapid degradation of waste material has been welldocumented by14,15 respectively. Suthar et al. 2008 (ref. 16) re-ported about the heavy metal accumulation ability of earth-worms in their tissues during vermicomposting. Numerousauthors, reviewed by Hughes et al. 1980 (ref. 17) and Beyer 1981(ref. 18) have reported that earthworms uptake and accumulateheavy metals such as Cd, Hg and Au in their tissues, while livingboth in contaminated and non-contaminated environments.19

Dai et al. (2004)20 reported that bioaccumulation of metals inworms is their ability to eliminate excess of metals. Metalsaccumulate in the chloragogen cells lying on the outerside ofthe earthworm's gut.21

Metallothionein (MT) is produced in earthworm's gut onexposure to heavy metals in their body.22 MT is a metal bindingubiquitous protein. It plays an important part in homeostasis ofmetal ion and redox chemistry within the cells.23 MT hasa binding capacity for several metals including Cu2+, Mn2+ andZn2+ themetalloid As3+.24 MT release depends upon a number offactors: duration of exposure, metal concentration, the presenceof specic metal ions and nature of feedstock.22

There are very few studies reported on the remediation ofindustrial wastes such as coal FA by the vermicompostingspecies and the comparison between the metal accumulationability of the different earthworm species. The experimentinvolves the exposure of the three earthworm species to thevermicomposting system consisting a mixture of FA and cow-dung (CD) in different ratios. The study emphasizes on (i)variations in biomass growth, count of earthworms and cocoonat different durations of vermicomposting. (ii) Accumulationand comparison of metals in the treatments and the threeearthworm species. (ii) Deriving relation between the metalaccumulation in the treatments and metal accumulation in theearthworm tissues based on regression equations. (iii) Bio-accumulation factor of metals in the earthworm species. (iv)Metallothionein (MT) protein production in the earthwormspecies on metal exposure from FA.

2. Materials and methods2.1. Sample collection

FA samples were collected from electrostatic precipitator ofChandrapura thermal power station (CTPS), Jharkhand, India(230073072� N and 860012056� E). Urine free, CD was collected fromlocal area in order to expedite the bioconversion process. Theearthworm species were collected from the vermiculture unit ofthe Vivekananda Institute of Biotechnology, Nimpith, Kolkata,India. The species selected for vermicomposting were twoepigeic earthworm species, E. fetida and E. eugeniae and an epi-endogeic earthworm species, L. rubellus. Mature (about 45–60


days old) healthy clitellated species of earthworms, comprisinga weight of about 350–420 mg were collected. The species werebrought to the laboratory in jute bags containing the feedmaterial.

2.2. Treatment design and vermicomposting system

Rectangular plastic bins used were of size: 45 cm (length), 25 cm(width), 30 cm (height). The bottom of each bin was providedwith twenty numbers of 100mmholes for aeration and drainagepurpose. CD and FA were air dried at room temperature. Thecombinations of FA and CD were thoroughly mixed and trans-ferred 3 kg of these substrates in each bin. Coal FA was mixedwith CD in different ratios along with the earthworm species asdescribed in Table 1. All the combinations were made in repli-cates of seven. The treatments were le for about 20 days beforethe inoculation of earthworms for thermal stabilization, initi-ation of microbial degradation and soening of substratematerial in shady regions. 36 earthworms were inoculated ineach bin. The optimum temperature and moisture content ofthe treatments were maintained at 25 � 2 �C and 60% respec-tively by sprinkling of water. The chemical and elementalcomposition were determined for the treatments at an intervalof 30 days (0, 30, 60, 90 days).

2.3. Cocoon, earthworm count and biomass growth

The number of cocoons and earthworms were periodicallycounted during vermicomposting by placing the samples onaluminium trays. Sieving was done in order to separate theearthworms from the white tiny cocoons. Biomass of theearthworms were measured at intervals using the methoddescribed by.25 The worms and cocoons were re-introduced intheir respective substrate mixtures carefully, aer counting andrecording the earthworm biomass.

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2.4. Chemical analysis and nutrient evaluation

The chemical parameters and nutrient content of the treat-ments were analyzed at an interval of 0, 30, 60 and 90 days. pH(1 : 2.5 w/v substrate : distilled water) was analyzed by a micro-processor based pH meter Esico model 1013. Electricalconductivity (EC, dS m�1) was analyzed in the ratio of 1 : 2 (w/v)by a digital conductivity meter (INSIF Electronics; IE-704). Totalphosphorous (TP) was determined by Olsen's method.26 Totalorganic carbon (TOC) was determined by a rapid dichromateoxidation method27 and Total Kjeldahl nitrogen (TKN) was ob-tained by the method employed by.28 Total potassium (TK)analysis was carried out on extracts prepared according to ref.29 and was estimated further by ame photometer.

2.5. Microbiological analysis

Bacterial and fungal colonies were isolated using serial dilutionpour plate techniques.30 Nutrient Agar (Hi-media) and RoseBengal Agar (Hi-media) were used to isolate bacterial and fungalcolonies respectively. The colonies obtained on the Petriplateswere counted with the help of a colony counter and the pop-ulation (total bacterial and total fungal count) were expressed interms of colony forming units (CFU g�1).

2.6. Heavy metal analysis

2.6.1. Vermicompost. The vermicompost was collectedfrom different treatments at different stages of the vermi-composting cycle. Heavy metal content in the treatment con-taining FA solely was determined by the wet digestion methodin a microwave digester (ETHOS One, Milestone MicrowaveDigester EPA 3546, Kawasaki, Japan) at 1000 W at 220 �C for 1 h0.2 g of homogenized sample was transferred into the Teonvessel with a mixture of acids, HNO3 + HF (5 : 1).31 For othertreatments (FA + CD), 0.5 g of sample was digested by adding25ml of nitric acid in a microwave digester and heated for 4 h at90–95 �C at 1000 W. Samples were then transferred into 100 mlpolyethylene bottles through lter paper Whatmann 42# andwere analyzed by Flame Atomic Absorption Spectrophotometer(FAAS) (GBC AVANTA 3000) to determine heavy metal concen-tration. The standard reference material (SRM) 2690, NationalInstitute of Standard and Technology, Ottawa, Canada (NIST)was used to check the validation of the results in the treatmentsEf1, Ee1, Lr1. Method validation in case of other treatments(vermicompost) was done using soil standard NIST-SRM 2709.

2.6.2. Earthworm tissues. Earthworms were properlyrinsed in distilled water and kept in Petri-dishes on moist lterpaper in the dark for 4 days at 20 � 2 �C for gut clearance. Thelter paper was changed repeatedly. The earthworms were keptin deep freeze at about �10 �C in order to prevent microbialdecomposition between collection and analysis. The earthwormtissues were oven dried at 80 �C for about 24 h.32 Katz andJennis33 method was applied to digest the earthworm tissues.The tissue samples were grounded inmortar pestle and burnt toash at 550 �C. The ash was placed in a test tube with 10 ml of55% nitric acid and was le overnight at room temperature fordigestion to start. The samples were heated at a temperature of

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60 �C for 2 h and further heated at 120 �C, till white fumes wereobtained. The samples were allowed to cool and the solutionswere ltered through whatmann lter paper 42# to 25 ml usingdeionized water. Heavy metals were estimated by FAAS (GBCAVANTA 3000). The accuracy of analytical procedures for metalestimation in earthworm tissues was done by Mussel tissueNIST-SRM 2976. All metal concentrations for SRMs were within10% of their certied values. The experiments were performedin compliance with the relevant laws and guidelines of IndianInstitute of Technology (Indian School of Mines), Dhanbad,Jharkhand, India.

2.7. Metallothionein radioassay

The radioassay of earthworm gut tissue was carried out by themethod employed by.34 The freeze killed worms were decolor-ized in 1.15% KCl solution and a homogenate was obtained byusing a glass Teon homogenizer.35 Hg203 was added to about1 ml of the homogenate. About 10% of 1 ml trichloroacetic acidwas added to a mixture of Hg and homogenate. The solutionwas made to stand for about 15 min to bring about theprecipitation of metallothienic lacking protein. Centrifugationof the entire mixture was done at 5000 rpm for about 10 min0.4 ml of the supernatant was assorted with about 4 ml ofscintillation uid i.e., cocktail. A Perkin Elmer 1220 Liquidscintillation counter from HESCO research laboratories, Indiawas used for determining the radioactivity. The radioactivitycontent was used as an estimate of MT and was expressed interms of nanomoles per g. The validation of the assay wasperformed by using the gel-ltration method.

2.8. Estimation of bioaccumulation factor (BAF)

Metal accumulation in earthworm tissues was evaluated withrespect to BAF. The factor was determined with respect to thebody tissues of earthworm species: E. fetida, E. eugeniae and L.rubellus and metal concentration in the respective substrates.Montouris et al. (2002)36 method was employed for estimationof BAF using the following formula:

BAF ¼ Cbiota/Csubstrate

where, Cbiota: metal concentration in earthworm tissues;Csubstrate: metal concentration in the respective treatments.

2.9. Statistical analysis

The statistical analyses were performed to determine mean andstandard deviation by the use of the XLSTAT package of MSExcel 2010. One way ANOVA was performed to determinesignicant differences between the biomass growths of earth-worms at different time intervals of vermicomposting process.Signicant differences in the number of earthworms andcocoons in different treatments were observed using Duncan'smultiple range test (ANOVA). Student's t test was performed toobtain signicant differences between mean of the metalconcentrations before (0th day) and aer (90th day) vermi-composting. Post hoc Tukey's test and Duncan's multiple rangetests were performed to observe variations in the chemical





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properties and nutrient content with different time intervalsand also to examine the signicant differences in the meanmetal concentrations of earthworm tissues among differenttreatments. Linear regression models were applied to deter-mine the degree of variance in concentration of metals in thesubstrates and the earthworms.

3. Results and discussion3.1. Variations in biomass, population of earthworms andcocoon production

The changes in biomass of earthworm species at different timeintervals of 0, 30, 60 and 90 days are depicted in Fig. 1. Anincrease in the trend of earthworm biomass of different specieswas observed to increase in the vermicomposting period.Maximum signicant (p < 0.05) increase in biomass wasobserved at 90th day of biological composting process. Poorbiomass growth of earthworms was observed in the treatmentcomprised solely of FA. This may be due to difficulty in propermovement of earthworms in sole FA treatments due to itscompact structure, thus reducing their reproduction rate. E.eugeniae showed a maximum increase in biomass followed by E.fetida and L. rubellus in FA + CD (1 : 3).

There was a considerable increase in the number of earth-worms and cocoons with increase in the duration of

Fig. 1 Biomass growth of the earthworm species during vermicompost(1 : 1); treatment 3: FA + CD (1 : 3); treatment 4: FA + CD (3 : 1); treatment� SD (n ¼ 7). Different letters in the line graphs with each colour indicdifferent time intervals at p < 0.05 according to Duncan's multiple range


vermicomposting (Fig. 2 and 3). E. eugeniae was observed tohave the most rapid growth with maximum number of cocoonproduction. E. eugeniae has high reproductive rates and has thecapacity to decompose large quantities of organic wastesrapidly.37 The treatments Ef2, Ef3 and Ef5 for E. fetida, Ee2, Ee3and Ee5 for E. eugeniae; Lr2, Lr3 and Lr5 for L. rubellus showedthe signicantly (p < 0.05) preeminent results in terms ofearthworm growth and cocoon production. Growth of earth-worms might be affected by suitable environment, availabilityof food and feed stock density of the earthworms.38,39 FA and CDmixtures (1 : 1) and (1 : 3) were found to be most compatiblemixtures for earthworm growth and cocoon production.

3.2. Variations in chemical properties and nutrient contents

3.2.1. pH and electrical conductivity (EC). A shi in pH canbe observed in Fig. 4a, 5a and 6a in treatments comprising E.fetida. Coal FA from CTPS had a slight basic pH. A slightincrease and nally a decrease in the pH values were observedin almost all the treatments (Fig. 4a, 5a and 6a). Similar resultsrelated to pH reduction during vermicomposting was observedby.40,41 Initial increase in pH may be due to alkaline humatesand aluminium complexes and its further reduction due toproduction of humic and fulvic acid during vermicompostingassociated decomposition of the substrates.42 Moreover, many

ing at different intervals. Treatment 1: FA alone; treatment 2: FA + CD5: CD alone. Error bars represent standard deviation; values are inmeanates significant differences in biomass growth of individual species attest (ANOVA).

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Fig. 2 Changes in the number of earthworms in the treatments at different intervals of vermicomposting process. (a) Eisenia fetida; (b) Eudriluseugeniae; (c) Lumbricus rubellus. Error bars represents standard deviation; values are inmean� SD (n¼ 7). Different letters in the bars with similarpattern indicates significant differences (p < 0.05) in the number of earthworms of the different treatments at the same time interval according toDuncan's multiple range test (ANOVA).

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intermediate organic acids are produced that result in reducingthe pH of the treatments.

An initial increase in EC values was observed in almost allthe treatments (Fig. 4b, 5b and 6b) upto 30–60 days. Decom-position of organic substances leads to the release of mineral

Fig. 3 Cocoon production by the earthworm species in the treatmentEudrilus eugeniae; (c) Lumbricus rubellus. Error bars represents standardpoints of the line graphs with individual colour represents significant diffa particular time interval of 0, 30, 60 and 90 days as per Duncan's multi

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salts like phosphates and ammonia, which may lead to aninitial increase in EC values. A sudden decline in EC values wasobserved aer this duration. Subsequent decrease in EC valuesat the end of vermicomposting may be due to precipitation ofmineral salts and volatilization of ammonia.43 Signicant

s during vermicomposting at different intervals. (a) Eisenia fetida; (b)deviation; values are in mean � SD (n ¼ 7). Different letters at the dataerences (p < 0.05) in the number of cocoon of different treatments atple range test (ANOVA).





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differences were found in EC values of the treatments at inter-vals of 0, 30, 60 and 90 days (ANOVA and Tukey's test; p < 0.05).

3.2.2. Total organic carbon, Kjeldahl nitrogen, phospho-rous and potassium. A signicant decrease in TOC wasobserved throughout the treatments. A signicantly higherdecline in TOC was noticed by E. eugeniae followed by E. fetidaand L. rubellus. A considerable decrease in TOC was observed intreatments Ef2 and Ef3 by E. fetida (Fig. 4c) and Ee2 and Ee3 byE. eugeniae (Fig. 5c) while, a subsequent less decrease in TOCwas observed by L. rubellus (Fig. 6c). Garg et al. (2006)44 alsoobserved the decline in TOC with duration of vermicomposting.Crawford 1983 (ref. 45) investigated that the decrease in TOCwas the resultant of carbon loss. Dominguez (2004)46 stated thatvermicomposting is a joint operation of earthworms andmicroorganisms involving fragmentation and homogenizationof the ingested material by the earthworms through muscularaction of their foregut. This in turn leads to addition of enzymesand mucus to the ingested material thereby, increasing thesurface area for microbial action. This biotic mutuality leads toloss of carbon in the form of CO2 during decomposition andmineralization of the substrates.47,48 The respiratory activity49

and high assimilation capacity of the earthworms furthercontributes to the decrease in TOC levels. An increase in thenumber of earthworms and consequently increase in microbialproliferation in the vermicomposts may have also resulted indecrease in TOC.49 Suthar and Singh (2008)50 reported that

Fig. 4 (a–f) Variations in chemical properties and nutrient content of diffthe vermicomposting process. Error bars represent standard deviation;pattern represent significant variation (at p < 0.05) in the chemical charatime intervals of the vermicomposting process according to Duncan's m


decline in TOC of the substrate mixture was followed bysubsequent increase in the N content.

TKN content increased signicantly in all the treatments withan increase in vermicomposting duration. In E. fetida, maximumincrease in TKN content was observed in Ef5 followed by Ef3 > Ef2> Ef4 > Ef1. High TKN values were observed at 90th day of vermi-composting in Ee5 (5.56%), Ee3 (5.54%) and Ee2 (5.24%) by E.eugeniae and Lr3 (3.43%) and Lr2 (1.9%) by L. rubellus (Fig. 4d, 5dand 6d). The increase in TKN valuesmight be due to loss in carbonand organic matter mineralization.51–53 Earthworms enhance Nlevel in the vermicompost by adding their excretory products,mucus, body uids and enzymes54,55 along with rapid microbialmineralization.38 Hartenstein and Hartenstein 1981 (ref. 56) re-ported that decrease in pHmay be an important factor in retainingN in the substrates as N is lost as ammonia at higher pH. Earth-worms pose a great impact on N transformations through modi-cations of the environmental conditions and their interactionwith microbes. They bring about mineralization of N, therebyresulting in conditions that favor nitrication, causing rapidconversion of ammonium-nitrogen into nitrates.9,41,57,58 However,Gaur and Singh (1995)59 and Kaviraj and Sharma (2003)52 reportedthat nal content of N in vermicompost is dependent on initial Npresent in the substrate and the extent of decomposition.

TP showed an increase in trend (Fig. 4e, 5e and 6e) duringdifferent time intervals of vermicomposting. High TP werefound in Ef3 (FA + CD; 1 : 3) and Ef5 (CD alone), comprising E.

erent treatments comprising Eisenia fetida at various time intervals ofvalues are in mean � SD (n ¼ 7). Different letters in the bars of similarcteristics and nutrient content of the individual treatments at differentultiple range ANOVA test and post hoc Tukey's test.

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Fig. 5 (a–f) Variations in chemical properties and nutrient content of different treatments comprising Eudrilus eugeniae at various time intervalsof the vermicomposting process. Error bars represent standard deviation; values are in mean � SD (n ¼ 7). Different letters in the bars of similarpattern represent significant variation (at p < 0.05) in the chemical characteristics and nutrient content of the individual treatments at differenttime intervals of the vermicomposting process according to Duncan's multiple range ANOVA test and post hoc Tukey's test.

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fetida. The maximum TP was noticed in treatments comprisingE. eugeniae. Ghosh et al. 1999 (ref. 60) revealed that vermi-composting has proved to be an effective technology inproviding good phosphorous nutrition. Earthworms have thecapability to convert phosphorous into soluble forms from theirinsoluble state.61 Satchell and Martein (1984)62 attributed thatincrease in P is the result of direct action of earthworms' gutenzymes and indirectly by stimulation of the microora.Edwards and Loy (1972)63 suggested that increase in TP duringvermicomposting may be possibly because of mineralizationand mobilization of P as a result of bacterial and faecal phos-phatase activity of earthworms.

TK showed signicant increase in trend throughout vermi-composting. Maximum increase in treatments were driven by E.eugeniae followed by L. rubellus and E. fetida (Fig. 4f, 5f and 6f).The treatments Ef3 and Ef5 of E. fetida, Ee5, and Ee4 of E.eugeniae and Lr5 and Lr3 of L. rubellus showed signicanthigher TK values in comparison to the other treatments. Itmight be due to conversion of complex organic matters tosimpler forms, that becomes more easily and readily availableto earthworm's gut,64 resulting in the higher availability ofpotassium. Garg et al. (2006)44 also observed higher TK values inthe vermicompost compared to the initial substrate mixture.They further reported that microbial ora inuences the level ofK and acid production by these microorganisms is an importantmechanism for solubilization of insoluble K.

4882 | RSC Adv., 2017, 7, 4876–4890

There was considerable improvement in the micronutrientcontent (NPK) in the treatments with increase in vermi-composting duration. However, E. eugeniae showed maximumenhancement of nutrient contents in FA + CD (1 : 3) and FA +CD (1 : 1). Similar results were reported by.12,65

3.3. Variations in microbiological fauna

High diversity in microbiological fauna was observed acrossthe treatments, except in FA alone, which may be due to slowgrowth and reproduction of earthworms in that particulartreatment. Higher microbial diversity was noticed in thetreatments comprising E. eugeniae followed by E. fetida and L.rubellus. Amongst the treatments, Ee3 showed the maximummicrobial diversity followed by Ee5 and Ef3 (Table 2).Maximum number of fungal strains were observed in thetreatment Ee3 (FA + CD; 1 : 3), followed by Ee2 (FA + CD; 1 : 2)(Table 3). Previous researchers have also reported that, thecombination of FA and organic amendment improves themicrobial functions.66,67 The maximum bacterial count (8.0 �105 CFU g�1) and fungal count (3.8 � 103 CFU g�1) wereobserved for the treatment Ee3. 11 bacterial strains wereobserved in the treatment FA + CD (1 : 3), comprising E.eugeniae, 9 bacterial strains in case of E. fetida and 7 bacterialstrains were observed in case of L. rubellus for the similartreatment.





Table 2 Variation in the microbial fauna of vermicomposted mixturesproduced by Eisenia fetida, Eudrilus eugeniae and Lumbricus rubellusa

TreatmentsBacterial count(value � 104 CFU g�1)

Fungal count(value � 102 CFU g�1)

Ef1 38* 10#

Ef2 60 20Ef3 70 34Ef4 48 18Ef5 66 27Ee1 55* 21#

Ee2 67 24Ee3 80 38Ee4 58 19Ee5 75 32Lr1 22* 8#

Lr2 48 20Lr3 64 25Lr4 38 19Lr5 56 22

a CFU: colony forming unit, *value� 102 CFU g�1, #value� 101 CFU g�1.

Table 3 Number of bacterial and fungal strains obtained in differenttreatments after vermicomposting

Treatments Bacterial strains Fungal strains

Ef1 2 0Ef2 8 2Ef3 9 2Ef4 3 1Ef5 5 1Ee1 3 0Ee2 6 2Ee3 11 3Ee4 4 1Ee5 7 1Lr1 0 0Lr2 5 1Lr3 7 2Lr4 3 0Lr5 4 1

Fig. 6 (a–f) Variations in chemical properties and nutrient content of different treatments comprising Lumbricus rubellus at various time intervalsof the vermicomposting process. Error bars represent standard deviation; values are in mean � SD (n ¼ 7). Different letters in the bars of similarpattern represent significant differences (at p < 0.05) in the chemical characteristics and nutrient content of the individual treatments at differenttime intervals of the vermicomposting process according to Duncan's multiple range ANOVA test and post hoc Tukey's test.

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3.4. Variations in metal concentration duringvermicomposting

3.4.1. Metal content in treatments. Table 4 depicts theconcentration of metals in the treatments before (0th day) andaer vermicomposting (90th day). There were signicantdifferences in the metal bioavailability of treatments from the


initial day of vermicomposting. E. fetida and E. Eugeniae havethe capability to bring about rapid degradation of FA and cow-dung mixtures. A signicant reduction in the concentration ofmetals such as Cu, Zn, Cr and Pb was observed in the treat-ments aer the vermicomposting. E. eugeniae was found to bevery effective in reduction of metal concentration for treat-ments Ee3 and Ee2. Reduction of Cr in the treatments Ef2 andEf3 by E. fetida was 38.28% and 58.82%, respectively. E.

RSC Adv., 2017, 7, 4876–4890 | 4883




Table 4 Changes in the metal concentration of the treatments comprising different earthworm species before (0th day) and after vermi-composting (90th day)a

Treatment

Cr Cu Zn Pb Ni Cd

0 day 90 day 0 day 90 day 0 day 90 day 0 day 90 day 0 day 90 day 0 day 90 day

Eisenia fetidaEf1 23.78

� 0.60c*20.51� 0.63a

66.54

� 1.68b*

58.22� 1.37a

98.56� 0.56a*

95.41� 1.44a

34.38� 0.48b*

30.13� 1.21a

23.51� 0.69b*

20.06� 1.10a

4.46� 0.23c*

2.51� 0.63b

Ef2 28.42� 0.77b*

17.54� 1.05b

56.45� 0.81c*

46.05� 1.39b

86.43� 2.18b*

71.78� 2.92b

32.57� 0.86c*

23.40� 1.75b

17.90� 0.47c*

11.31� 0.60c

3.38� 0.39d*

1.65� 0.03d

Ef3 18.60� 0.74d*

7.66� 0.05d

35.75� 0.89d*

15.43� 2.55c

66.85� 1.80c*

50.79� 0.00d

24.98� 2.04d*

14.38� 0.00c

12.69� 1.1d*

9.57� 0.05c

2.55� 0.14e*

1.25� 0.05e

Ef4 33.99� 0.62a*

17.24� 0.43c

67.93� 0.85a*

38.90� 1.05a

98.09� 0.46a*

54.45� 1.58c

35.23� 0.40a*

22.81� 1.08b

28.7� 0.72a*

16.2� 0.90b

4.87� 0.02b*

2.43� 0.11c

Ef5 5.66� 0.22e*

2.31� 0.03e

10.50� 0.29e*

2.31� 0.03d

12.67� 0.76d*

8.34� 0.08e

1.15� 0.04e*

0.57� 0.05d

0.84� 0.02e*

0.55� 0.02d

6.47� 0.18a*

3.29� 0.14a

Eudrilus eugeniaeEe1 23.86

� 0.44b*17.94� 0.67a

66.16� 0.77a*

20.47� 0.63c

98.93� 1.58a*

90.71� 1.46a

34.36� 0.59a*

31.07� 0.79a

23.87� 1.00a*

18.28� 1.08a

4.58� 0.14b*

2.59� 0.07a

Ee2 28.35� 0.21a*

15.43� 0.13b

56.59� 1.75b*

45.96� 1.70a

85.36� 0.51c*

70.42� 3.43b

32.71� 0.78b*

16.39� 0.41b

17.74� 0.28b*

10.52� 0.08b

3.33� 0.16c*

2.10� 0.10b

Ee3 18.65� 0.16c*

7.60� 0.03d

34.83� 2.15c*

14.36� 0.38c

69.77� 1.63c*

32.70� 2.70d

24.23� 0.84c*

14.05� 0.43c

12.65� 0.06c*

3.55� 0.16c

2.59� 0.05d*

1.76� 0.04d

Ee4 23.77� 0.11c*

12.54� 0.61c

54.77� 0.61b*

27.33� 0.20b

88.58� 0.03b*

56.74� 0.14c

33.25� 0.01b#

13.11� 0.15c

28.5� 1.46a*

21.58� 0.09a

6.92� 0.01b*

3.54� 0.001a

Ee5 9.57� 0.02d*

7.06� 0.08e

10.73� 0.23d*

5.99� 0.12d

12.83� 0.72d#

4.47� 0.15e

2.67� 0.12d*

1.48� 0.04d

1.58� 0.00d*

1.21� 0.03d

6.80� 0.68a*

2.54� 0.19a

Lumbricus rubellusLr1 23.83

� 0.57c*22.13� 1.62a

64.96� 1.44b*

58.24� 1.73a

97.45� 2.64a*

94.92� 0.60a

34.25� 0.87a*

34.13� 1.47a

23.49� 0.20b*

20.59� 0.49a

4.55� 0.04c*

2.49� 0.23b

Lr2 28.09� 0.44b*

17.93� 0.59b

56.50� 2.33c*

50.42� 2.70a

84.30� 1.52b*

72.62� 0.59b

32.62� 0.35b*

22.29� 0.50c

17.52� 0.53c*

13.82� 0.21b

3.31� 0.06d*

1.45� 0.08c

Lr3 18.63� 0.38d*

15.56� 0.12b

34.72� 4.57d*

16.78� 0.53c

67.15� 2.81c*

52.47� 1.75c

24.16� 0.66c*

15.39� 0.86d

12.63� 0.01d*

7.52� 0.00c

4.60� 0.03c*

2.84� 0.00b

Lr4 33.61� 1.34a*

22.50� 0.63a

68.35� 2.26a*

45.58� 0.59c

98.35� 3.39a*

64.19� 0.70c

43.67� 0.01d*

32.23� 0.15b

24.30� 0.07b*

11.47� 0.20b

2.54� 0.00d*

1.24� 0.20c

Lr5 5.68� 0.03e*

2.54� 0.33c

10.74� 0.28e*

6.44� 0.16d

12.72� 0.26d*

8.31� 0.12d

1.14� 0.00e*

0.82� 0.05e

2.85� 0.01e*

2.54� 0.02d

7.21� 0.38a*

2.7� 0.40b

a Values are in mean � SD (n ¼ 7). Different letters in blue colour indicate signicant differences (at p < 0.05) in the mean metal concentrations ofthe different treatments before vermicomposting (0 day), according to Duncan's multiple range test (ANOVA) and post hoc Tukey's test. Differentletters in purple colour represent signicant differences (at p < 0.05) in the mean metal concentrations of the different treatments aervermicomposting (90th day) as per Duncan's multiple range test (ANOVA) and Tukey's test. *Signicant differences (p < 0.05) between means ofmetal concentrations in the substrates before (0th day) and aer vermicomposting (90th day) (student's t-test).

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eugeniae reduced Cr concentration up to 59.25% while,L. rubellus up to 16.48%. Cu concentration was reduced tomore than 50% in Ef3, Ee3 and Lr3 by the three species ofearthworms. Signicant reduction in Zn and Pb were noticedby E. fetida and E. eugeniae. E. fetida reduced Cd upto 50.98%,showing an affinity towards Cd while, L. rubellus and E.eugeniae reduced Cd upto 38.26% and 32% respectively. E.eugeniae showed remarkable results by reducing Ni upto72.33% for Ee3. Gupta et al. 2005 (ref. 13) and Bhattacharyaand Chattopadhyay 2006 (ref. 68) demonstrated the role ofearthworms in mitigating the toxicity of FA. Niyazi et al. 2014(ref. 69) observed a decrease in heavy metal concentrations ofthe nal vermicompost on inoculation of earthworms.70 Thesignicant reduction in metal concentration of the compostduring vermicomposting has also been reported by.71,72

4884 | RSC Adv., 2017, 7, 4876–4890

Goswami et al. (2016)73 reported a steady decline in the totalmetal content in the vermicomposted products suggestingthat, a considerable amount of metals may be accumulated bythe earthworms.

Earthworms play a vital role in immobilization of metals bythe process of metal detoxication via vermiremediation tech-nology. This occurs due to two probable reasons. Firstly theearthwormmediated biodegradation process increases the levelof humic fractions, that strongly immobilize the formation ofstable metal–humus complexes74 and secondly, the metalchelating metallothionein proteins, present in the gut ofearthworms bind metals as per the pathway described by.75

Improvement in the degree of humication indicates theformation of highly stable aromatic compounds, which havegreat potential for metal sequestration.74,76





Fig. 7 Fluctuations in metal concentrations of the earthworm tissues in different treatments before and after vermicomposting. Treatment 1: FAalone; treatment 2: FA + CD (1 : 1); treatment 3: FA + CD (1 : 3); treatment 4: FA + CD (3 : 1); treatment 5: CD alone. Different letters in blackcolour represents significant differences (p < 0.05) in mean metal concentrations of Eisenia fetida in different treatments as per ANOVA andTukey's test; different letters in blue colour represents significant differences (p < 0.05) in mean metal concentrations of Eudrilus eugeniae indifferent treatments as per ANOVA and Tukey's test; values are in mean � SD (n ¼ 7). Different letters in purple colour represents significantdifferences (p < 0.05) in mean metal concentrations of Lumbricus rubellus (ANOVA; Tukey's test).

This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 4876–4890 | 4885

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Table 5 Linear regression model, determination coefficient andsignificance levels with Cr, Cu, Zn, Pb, Ni, Cd concentration in Eiseniafetida, Eudrilus eugeniae and Lumbricus rubellus as predictor variables.Metal concentration in the substrates served as dependent variables.Models consisting of significant predictor variables are only showna

Model R2 Signicance

Eisenia fetida[Cr]S ¼ 24.72 � 1.94*[Cr]Ef 0.53 0.17[Cu]S ¼ 48.16 � 0.02*[Cu]Ef 0.08 0.64[Zn]S ¼ 60.74 � 0.53*[Zn]Ef 0.012 0.86[Pb]S ¼ 17.00 + 0.25*[Pb]Ef 0.004 0.92[Ni]S ¼ 2.61 + 2.18*[Ni]Ef 0.25 0.40[Cd]S ¼ 4.40 � 0.81*[Cd]Ef 0.60 0.13

Eudrilus eugeniae[Cr]S ¼ 17.04 � 0.36*[Cr]Ee 0.12 0.57[Cu]S ¼ 36.16 � 1.00*[Cr]Ee 0.13 0.56[Zn]S ¼ 51.46 � 0.03*[Cr]Ee 6.11 � 10�5 0.99[Pb]S ¼ 13.50 � 0.22*[Pb]Ee 0.008 0.89[Ni]S ¼ 11.11 � 0.01*[Ni]Ee 2.75 � 10�5 0.99[Cd]S ¼ 2.53 � 0.004*[Cd]Ee 5.07 � 10�5 0.99

Lumbricus rubellus[Cr]S ¼ 34.49 � 2.099*[Cr]Lr 0.12 0.57[Cu]S ¼ 44.19 � 0.88*[Cu]Lr 0.07 0.68[Zn]S ¼ 59.11 + 0.09*[Zn]Lr 0.00 0.98[Pb]S ¼ 24.04 � 0.91*[Pb]Lr 0.03 0.80[Ni]S ¼ 13.43 � 0.33*[Ni]Lr 0.06 0.70[Cd]S ¼ 1.84 + 0.06*[Cd]Lr 0.02 0.84

a [M]S: metal concentration in the substrates (mg kg�1), [M]Ef: metalconcentration [Cr, Cu, Zn, Pb, Ni, Cd] in the tissues of Eisenia fetida(mg kg�1), [M]Ee: metal concentration in Eudrilus eugeniae (mg kg�1),[M]Lr: metal concentration in Lumbricus rubellus. Regression at 95%condence level.

Table 6 Bioaccumulation factor (BAF) of metals in Eisenia fetida, Eudrilu

Treatments Cr Cu Zn

Eisenia fetidaEf1 0.18 � 0.008d 0.15 � 0.00c 0.03 � 0.0Ef2 0.40 � 0.004c 0.19 � 0.007e 0.12 � 0.0Ef3 1.21 � 0.03b 1.29 � 0.05b 0.34 � 0.0Ef4 0.21 � 0.02d 0.38 � 0.01d 0.14 � 0.0Ef5 3.91 � 0.04a 3.68 � 0.01a 0.54 � 0.0

Eudrilus eugeniaeEe1 0.42 � 0.02d 0.29 � 0.002d 0.05 � 0.0Ee2 1.10 � 0.08c 0.22 � 0.04d 0.31 � 0.1Ee3 2.44 � 0.02b 1.33 � 0.05b 0.63 � 0.0Ee4 1.19 � 0.22c 0.61 � 0.10c 0.20 � 0.1Ee5 1.49 � 0.08a 2.43 � 0.12a 1.54 � 0.0

Lumbricus rubellusLr1 0.34 � 0.02d 0.08 � 0.004d 0.02 � 0.0Lr2 0.51 � 0.02c 0.10 � 0.004d 0.09 � 0.0Lr3 0.69 � 0.03b 1.09 � 0.02a 0.28 � 0.0Lr4 0.33 � 0.07d 0.35 � 0.05c 0.10 � 0.0Lr5 3.44 � 0.003a 0.84 � 0.00b 0.31 � 0.0

a Values are in mean � SD (n ¼ 7). Different letters in the same column reearthworm species in different treatments.

4886 | RSC Adv., 2017, 7, 4876–4890

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3.4.2. Metal content in earthworm tissues. The metalconcentrations (Cr, Cu, Zn, Pb, Ni, Cd) in the tissues of E. fetida,E. eugeniae and L. rubellus before (0th day) and aer vermi-composting (90th day) have been depicted in Fig. 7. The metalaccumulation capacity of the earthworm species was largelyaffected by the metal concentration. Signicant differences (p <0.05) were observed in the metal concentrations of earthwormtissues of treatments before and aer vermicomposting. E.eugeniae showed the maximum accumulation of Cr in theirtissues for Ee3. Wang et al. 2009 (ref. 77) reported E. eugeniae tobe an important bio-accumulator and bioindicator of environ-mental contamination, comprising of persistent pollutants likeheavy metals. Cu was efficiently accumulated by all the threespecies in the treatments: Ef3, Ef4, Ee3, Ee4, Lr3 and Lr4, indi-cating high affinity of these worms towards Cu. Higher concen-tration of Zn was accumulated by E. eugeniae in Ee2. Morgan andMorgan 1999 (ref. 78) and Kizilkaya 2005 (ref. 79) have reportedthat bioaccumulation may be strongly inuenced by physico-chemical and edaphic interactions, including factors such asorganic matter content, and C-to-N ratio. The variability in bio-accumulation of metals in earthworm tissues may depend uponinterspecic differences in chemical species requirements,physiological and morphological characteristics,80 type ofspecies, ecological category of earthworm species, season andseveral other factors.81 Metal accumulation ability of E. eugeniaeduring vermicomposting has been documented by severalauthors.14,82,83 Suthar 2009 (ref. 55) and Yadav and Garg (2011)84

observed the metal accumulation ability of E. fetida. The poten-tial of metal removal by L. rubellus has been observed by.67,85

Earthworm species (E. fetida, E. eugeniae and L. rubellus) havean inherent tendency to accumulate heavy metals in their gut,using low molecular weight, metal chelating, cysteine rich MT

s eugeniae and Lumbricus rubellusa

Pb Ni Cd

001d 0.13 � 0.008d 0.18 � 0.02c 1.09 � 0.003c05c 0.28 � 0.002c 0.48 � 0.004b 1.42 � 0.01b3b 0.65 � 0.03b 0.57 � 0.51b 3.12 � 0.05a1c 0.11 � 0.006d 0.28 � 0.02c 1.10 � 0.02b07a 4.65 � 0.24a 3.91 � 0.13a 0.56 � 0.009d

0d 0.15 � 0.003c 0.26 � 0.01d 1.11 � 0.06d4b 0.74 � 0.07b 0.89 � 0.07b 1.69 � 0.03c3b 0.89 � 0.10b 3.31 � 0.30b 2.74 � 0.62b4c 0.57 � 0.16d 0.34 � 0.002a 1.25 � 0.046a 1.93 � 0.03a 1.35 � 0.36c 2.10 � 0.45c

0c 0.10 � 0.004e 0.06 � 0.002b 1.18 � 0.004e0b 0.15 � 0.01c 0.54 � 0.00d 3.73 � 0.05a09a 0.47 � 0.02b 1.18 � 0.02a 2.64 � 0.19b06b 0.03 � 0.02d 0.28 � 0.04c 4.39 � 0.03d1a 2.38 � 0.09a 0.96 � 0.05b 2.01 � 0.10c

present signicant differences (at p < 0.05) in bioaccumulation factor of





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proteins,86 present in the chloragogenous tissues of their gut. MTregulates the bioavailability detoxication dynamics of essentialand non-essential metals in the earthworm guts.87 Earthwormshave two concurring metal binding mechanisms as per theprevious literature.88 Firstly, the metals are retained in insolublecalcium phosphate granules or chloragosomes.86 Due to this,metals remain insoluble and cannot inuence the normal bio-chemical process in the cytoplasm.86 Later, these insolublemetals are chelated by the sulphur donating ligands of MTs andare carried to the chloragogenous tissues of the earthworms'intestines, where they are neutralized.88 This metal pathway hasbeen supported with daunting proofs in a recent report of.75

Fig. 8 (a–c) Elution profiles of sephadex G-75, Hg203 treated acid fractirubellus tissue. (d–f) Variation in the level of metallothionein (nmol g�1) inexposure to FA and CDmixtures during the process of vermicomposting.


A relationship has been derived between the metal concen-tration of the treatments consisting of FA and CD in differentratios and the metal concentration in the respective earthwormtissues. A comparison of regression models of E. fetida, E.eugeniae and L. rubellus have been shown in Table 5. Metalconcentration in earthworm species as predictor variablesexplained the variation in metal concentrations of Cr, Cu, Zn,Pb, Ni and Cd.

3.5. Bioaccumulation factor

Bioaccumulation factor, evaluated with respect to the metalconcentration in the treatments and earthworms is given in

onated supernatant of Eisenia fetida, Eudrilus eugeniae and Lumbricustissues of Eisenia fetida, Eudrilus eugeniae and Lumbricus rubellus on

Values are inmean� SD (n¼ 7). Error bars represent standard deviation.

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Table 6. BAF values greater than 1 indicates higher metalconcentration in earthworm tissues than that of the substrate.36

In case of treatments, comprising E. fetida, BAF was generallyfound to be below 1, except for Ef3 and Ef5 in case of Cr and Cu,Ef5 in case of Pb and Ni, Ef1 and Ef4 for Cd. In case of E.eugeniae, the highest value of BAF was found for Ni, (3.31) inEe3, indicating higher metal concentration in earthwormtissues as compared to other treatments. High BAF values forCd were found in Ee3 followed by Ee5 > Ee2 > Ee4. Treatmentscomprising L. rubellus, Lr4 showed the highest BAF value for Cdfollowed by Lr2 > Lr3 > Lr5 > Lr1, while, treatment Lr3 showedthe highest BAF value for Ni.

Bioaccumulation is a direct biological measure of metalbioavailability as it measures the actual amount of metal uptakeby the earthworm over the duration of earthworm exposure tothe substrate.89 BAF for metals vary widely with the specicmetal, the specic organism, age of the specic organism andspecic exposure circumstances determined. Different metalshave different chemical properties and different earthwormspecies have their specic physiological behaviors underdifferent life stages.89 A combination of these factors contrib-utes to the variation in metal bioaccumulation as evaluated byBAFs.

3.6. Variation in metallothionein content

The sephadex G-75 elution prole of Hg203 incubated and acidfractionated supernatant of E. fetida, E. eugeniae and L. rubelluswere demonstrated on radioactive absorbance of 254 nm peak(Fig. 8a–c). This determines the accuracy of MT radioassay.Exposure to heavy metals induces synthesis of MT isoforms inearthworm intestine,20 which can detoxify metal ions (As, Hg, Si,Al, Fe, etc.) to a considerable extent.22 Low molecular mass, MTproteins with very high cysteine content,90 have a high affinitytowards certain trace metal ions like copper, cadmium andzinc.91 The cysteine residues form ligands using thiolate bondswith metal atoms.91 Assessment of MT radioactivity is necessaryinorder to characterize the mechanism of metal accumula-tion.92,93 There was a substantial increase in MT levels in E.fetida, E. eugeniae and L. rubellus (Fig. 8d–f). Maximum MTcontent was observed in treatment Ee3 (415 nmol g�1) followedby Ef3 (380 nmol g�1), and Lr3 (305 nmol g�1). The trend for theelution prole of sephadex G-75 and theMT content showed thefollowing order: E. eugeniae > E. fetida > L. rubellus. MT contentwas maximum in E. fetida for the treatment Ef3 (FA + CD; 1 : 3),while in E. eugeniae, MT content was highest for Ee2 (FA + CD;1 : 1). The treatment Lr3 showed the maximum MT content forL. rubellus. Goswami et al. (2016)73 also observed signicantlyhigh level of MT in E. fetida on exposure to metal loaded teafactory coal ash mixtures.

4. Conclusion

The study concludes that earthworms holds an important rolein processing FA into organic rich manure free from metals.Earthworm species played an important role in enhancing thenutrient content of FA and CD associated mixtures. E. eugeniae

4888 | RSC Adv., 2017, 7, 4876–4890

showed better overall metal removal efficiency from the treat-ments as compared to E. fetida and L. rubellus. E. eugeniaeshowed more affinity towards accumulation of Cr, Cu, Zn, Cdand Pb followed by E. fetida. MT content showed synchrony withthe level of metal accumulation in earthworms. The mosteffective results in terms of nutrient content and metal reme-diation was shown for FA + CD (3 : 1) mixture. Thus, vermi-composting of coal FA appears viable mostly at lowincorporation rates ranging from 33% to 50%. These applica-tion rates may not be a serious challenge as the heavy metalsemanating from FA composting can be efficiently remediated bythe earthworms thus, falling within permissible limits outlinedfor other waste. The regression equations derived relationsbetween metal concentration in the treatments and wormtissues of respective substrates, thus supporting the importanceof uptake of these metals by the different species of earth-worms. Studies related to the impact of metals on the earth-worm's cell structure and genes should be done in order tosafely and properly execute the vermicomposting process ofcoal FA.

Acknowledgements

The authors are grateful to the Department of EnvironmentalScience and Engineering and Central research facility, IndianInstitute of Technology (Indian School of Mines), Dhanbad forproviding research facilities.

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vermicomposting of coal fly ash using epigeic and epi

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